基于microRNA探讨电针延缓HepG2裸鼠荷瘤生长趋势效应的初步机制

Study on Retarding Growth Trend Effect of Electroacupuncture in HepG2 Hepatocellular Carcinoma in Nude Mice based on microRNA

目的:基于miRNAs测序技术探讨电针对HepG2裸鼠荷瘤生长趋势延缓效应的初步作用机制。方法:将20只SPF级裸鼠随机分成电针组(EG)和对照组(CG),均采用皮下接种HepG2-Luc细胞悬液的方法进行肝癌模型造模。接种1周后使用小动物活体成像仪确认造模成功,EG组裸鼠开始行电针干预,30 min/次,每天1次;CG组裸鼠仅采用相同的方法进行固定;均连续操作14 d。干预期间观察并记录荷瘤体积变化,计算生长趋势;并在干预结束后,使用RNA-seq高通量测序分析,筛选肿瘤中差异表达的miRNAs,运用生物信息学方法进行靶基因预测。结果:(1)EG组在荷瘤体积变化趋势延缓方面有一定优势,与CG组差异有统计学意义(P<0.05)。(2)经测序分析筛选出24个荷瘤组织两组间差异表达的miRNAs,其中上调20个,下调4个。采用GO及KEGG聚类分析,显示表达水平有差异的miRNAs,其靶基因主要参与细胞组成的蛋白代谢和肿瘤炎症相关通路。(3)综合表达量、差异倍数及检索现有文献的基础上,继续选择其中15个miRNAs进行qPCR验证,显示miR-144-3p、miR-486-5p、miR-487b-3p及miR-451a组间表达水平有显著的统计学差异(P<0.05)。结论:电针具有一定的延缓HepG2肝癌裸鼠荷瘤生长趋势作用,其机制可能与上述4个经验证的miRNAs所参与的蛋白代谢通路、肿瘤相关通路及MAPK信号通路相关,但仍需进一步实验明确其中极为复杂的调控网络。

Objective:To verify the effect of electroacupuncture in retarding the growth trend of HepG2 hepatocellular carcinoma in nude mice based on miRNA.Methods:20 BALB/c-nude mice in SPF level were randomly divided into electroacupuncture group(EG)and control group(CG),10 mice in each group.EG and CG group were inoculated with HepG2-Luc cell suspension for modeling.After confirmation of successful modeling,interventions of EG were given daily for 30 minutes each,and CG mice were fixed daily using the same method as EG,both for 14 consecutive days.Observing the trend of subcutaneous tumor volume by directly measuring or via small animal imaging system.Post-intervention,differentially expressed miRNAs were analyzed by RNA-Seq high-throughput sequencing method,and the target genes of differentially expressed miRNAs were selected to be analyzed with combination of biological information.Results:(1)The EG had an advantage in delaying the trend of tumor volume growth,with a statistically significant difference(P<0.05).(2)After sequencing and analysis,a total of 24 differentially expressed miRNAs were screened in the tumor tissues of the EG and CG,including 20 up-regulated and 4 down-regulated miRNAs.Analyzed by Go and KEGG,the target genes of miRNAs which were influenced by electroacupuncture mainly related with protein metabolism and tumor inflammation-related pathways.(3)Based on the expression,folding difference and searching of current literatures,15 miRNAs were selected for qPCR validation,showing statistically significant differences in expression between miR-144-3 p,miR-486-5 p,miR-487 b-3 p and miR-451 a groups(P<0.05).Conclusion:Electroacupuncture has the effect in retarding the growth trend of HepG2 hepatocellular carcinoma in nude mice.Its mechanism may be highly relevant to the protein metabolic pathways,tumor-associated pathways and MAPK signaling pathways in which the 4 validated miRNAs mentioned above are involved.